Pre-Formed Fibrils, WT α-Synuclein (α-syn-WT PFFs)

Pre-formed fibrils (PFFs) of wild type (WT) alpha-synuclein.

Alpha-synuclein (α-synuclein) is an intrinsically disordered protein of unknown function that is linked to both familial and sporadic cases of Parkinson's disease, where it forms the major constituent of the pathological Lewy body aggregates. These fibrils can be used for disease modelling since similar PFFs have been shown to seed endogenous α-synuclein inclusions in healthy cells.

SAFETY: Wear gloves, disposable sleeves, and a second pair of gloves when handling PFFs. Dispose of pipette tips and microfuge tubes in a container of 1% SDS. Wear protective headphones while sonicating fibrils, and decontaminate probe and spills with 1% SDS.

Preparation:

Obtain stock PFFs from -80 freezer (stocks contain 10uL of 5mg/mL fibrils, and contain either WT or mutant α-synuclein; WT is used for most applications).

Place PBS, 1% SDS and a P1000 in TC hood. Don sleeves and second pair of gloves.

Pipette PBS into PFF aliquot depending on desired concentration (commonly 490uL is added to give a concentration of 0.1mg/mL) and place used tip in SDS.

Remove outer gloves when leaving hood.

Sonication:

Attach 1/8Ó microtip to sonicator (if not previously attached) and use wrench to tighten

Clean the probe and the door of the enclosure with 70% EtOH.

Set the sonicator to the following settings: 20% amplitude; 30 seconds; 1 second on, 1 second off

Don a second pair of gloves.

Place fibril tube in a tube rack in the sonicator enclosure

Adjust the stand height such that the probe tip is submerged in the solution (about halfway between the solution surface and tube bottom), and adjust the rack so that the probe is in the middle of the tube.

Remover outer gloves, close enclosure, don protective headphones, make sure Do Not Enter sign is on door, and press start.

If bubbles form or the probe loosens during sonication, stop the sonication, don a second pair of gloves, and adjust as necessary.

After sonication, don a second pair of gloves, remove and close the PFF tube, which is now ready for use.

Clean the probe and anything the PFFs touched with 1% SDS. Clean again with 70% EtOH.

In cell culture, these sonicated PFFs are typically used at 1% vol/vol (or 10uL/mL of media).